Inhibition mechanism of the chloride channel TMEM16A by the pore blocker 1PBC

Abstract

TMEM16A, a calcium-activated chloride channel involved in multiple cellular processes, is a proposed target for diseases such as hypertension, asthma, and cystic fibrosis. Despite these therapeutic promises, its pharmacology remains poorly understood. Here, we present a cryo-EM structure of TMEM16A in complex with the channel blocker 1PBC and a detailed functional analysis of its inhibition mechanism. A pocket located external to the neck region of the hourglass-shaped pore is responsible for open-channel block by 1PBC and presumably also by its structural analogs. The binding of the blocker stabilizes an open-like conformation of the channel that involves a rearrangement of several pore helices. The expansion of the outer pore enhances blocker sensitivity and enables 1PBC to bind at a site within the transmembrane electric field. Our results define the mechanism of inhibition and gating and will facilitate the design of new, potent TMEM16A modulators.

Structural basis for the activation of the lipid scramblase TMEM16F

“…We constructed mutants of this flexible residue with the aim to rigidify this region and investigated the consequence of the mutation on activation. The mutation of the equivalent position (i.e., Gly 510) in TMEM16A to a rigid proline, investigated in a previous study, has shown a pronounced right-shift of the EC 50 of Ca 2+ , reflecting a strong destabilization of the open state 42 . The concomitant weakening of its inhibition by an open-channel blocker, which binds to a close-by pocket that is formed in the active state, further underlines the restriction of conformational rearrangements by the mutation 42 .…”

The pharmacology of the TMEM16A channel: therapeutic opportunities

“…The determination of the TMEM16A structure and elucidation of the gating mechanisms can now facilitate the rational drug design of small-molecule modulators acting on specific domains of the channel. Some of the recently identified small-molecule-binding sites involve the TM6 gate [11][12][13], and it remains to be determined whether molecules can be generated to selectively act on other functionally important regions of the channel. Like numerous other established drug targets, including ion channels [e.g., Ca v channels, ATP-sensitive K + (K ATP ) channels], TMEM16A is expressed in more than one tissue type.…”

“…The binding sites for TMEM16A modulators remain largely undefined, although a combination of single point mutagenesis, electrophysiology, and molecular modelling led to the identification of putative binding sites for synthetic (e.g., A9C [11]) and endogenous (PIP 2 [9,10]) modulators (Figure 1). The structures of the TMEM16F [12] and TMEM16A [13] in the presence of either 1-hydroxy-3-(trifluoromethyl)pyrido[1,2-a]benzimidazole-4-carbonitrile (1PBC) [13], a potent inhibitor with antitumoral properties, or niclosamide have been obtained. The molecules bind to the extracellular-proximal end of TM6 (steric gate) of TMEM16F and TMEM16A [12,13], a region that also encompasses the A9C-binding site in TMEM16A [11].…”

“…The structures of the TMEM16F [12] and TMEM16A [13] in the presence of either 1-hydroxy-3-(trifluoromethyl)pyrido[1,2-a]benzimidazole-4-carbonitrile (1PBC) [13], a potent inhibitor with antitumoral properties, or niclosamide have been obtained. The molecules bind to the extracellular-proximal end of TM6 (steric gate) of TMEM16F and TMEM16A [12,13], a region that also encompasses the A9C-binding site in TMEM16A [11]. Whether synthetic small molecules can interfere with the electrostatic gate or binding sites for lipids such as PIP 2 remains to be determined.…”

“…Our detailed understanding of CaCC physiology and pharmacology has long been impeded by not knowing the channel's molecular structure. The identification of TMEM16A as a gene encoding CaCC [3][4][5] electrified the ion channel field and triggered a wealth of investigations leading to the determination of the TMEM16A 3D structure [6][7][8], the identification of binding sites for endogenous [9,10] and synthetic [11][12][13] ligands or natural products [14,15], and the elucidation of the role of the channel in a range of cell types [1,2,16]. While research efforts enabled the discovery of potent test compounds (inhibitors and activators), no therapeutic drugs acting on TMEM16A have yet reached use in clinical practice.…”